Improvement of crop plants for a variety of traits, including disease and pest resistance, and grain quality improvements such as oil, starch or protein composition, can be achieved by introducing new or modified genes (transgenes) into the plant genome. Expression of genes, including transgenes, is in general controlled by the promoter through a complex set of protein/DNA and protein/protein interactions. Promoters can impart patterns of expression that are either constitutive or limited to specific tissues or times during development. There are limitations in the types of expression achievable using existing promoters for transgene expression. One limitation is in the expression level achievable. It is difficult to obtain traits that require relatively high expression of an introduced gene, due to limitations in promoter strength. A second limitation is that the pattern of expression conferred by the particular promoter employed is inflexible in that the same promoter-dependent pattern of expression is conferred from generation to generation.
For imparting certain traits, it is desirable to have the ability to regulate the trait-conferring transgene expression differently in successive generations. One example would be a trait that has a side effect of being detrimental to seed viability, but which is desired in a grain product. For example, one can envision using plant seeds to produce proteins, starches, or other substances in grain (product seeds not used for further planting) which are detrimental to seed viability. In this case it would be desirable to carry the trait-conferring transgene in an inactive state in breeding lines up until the time of grain production. Then a new type of expression system is required to activate the trait gene in the grain.
Another limitation in the current methods of transgene expression is an inability to coordinately regulate multiple transgenes in transgenic crops. Multiple copies of the same promoter, directing coordinate regulation of multiple genes, can lead to gene inactivation through repeat induced gene silencing (Ye and Signer, 1996, Proc. National Acad. Sci. 93:10881-10886) or other means of cosuppression. Thus, a means of coordinately regulating multiple transgenes is desirable.
Transcriptional activation is primarily mediated through transcription factors that interact with enhancer and promoter elements. Binding of transcription factors to such DNA elements constitutes a crucial step in transcriptional initiation. Structural and finctional analyses of transcription factors revealed that many of these proteins have a modular protein structure, i.e., they are often modular, made up of a specific DNA-binding domain and a separate and independently acting activation domain. Researchers have found that heterogeneous domains can be combined, the resultant composite activators being functional in mammalian cells. An example of such an activator is the protein produced by fusion of the Gal4 DNA-binding domain with the activation domain of VP16.
Each transcription factor binds to its specific binding sequence in a promoter and activates expression of the linked coding region through interactions with coactivators and/or proteins that are a part of the transcription complex. A DNA binding domain and an activation domain derived from different proteins can be linked to produce a chimeric transcription factor. One transcription factor that has been studied is the yeast transcription factor Gal4 which is composed of a DNA binding domain and an activation domain. Native Gal4, a protein of 881 amino acids, is a transcriptional activator of genes required for galactose catabolism in the yeast S. cerevisiae. The protein binds specifically to the upstream activating sequence called UAS.sub.G and activates transcription of the divergently transcribed genes GAL1 and GAL 10.
A two component transcription factor/target promoter system could be used to address the above limitations of transgene expression with existing promoters. For example, a chimeric transcription factor comprising the Gal4 DNA binding domain could be used as the basis of a two component gene expression system. In fact, chimeric transcription factors containing the Gal4 DNA binding domain and various activation domains have been successfully used in plant cells in transient assays, but not in stable transformants.
The yeast Gal4 DNA binding domain fused to one or two of its own activation domains was able to activate expression of a chloramphenicol acetyl transferase (CAT) reporter gene, with Gal4 binding sites in the promoter, in a transient assay in tobacco protoplasts (Ma et al., 1988 Nature 334, p 631-633). The expression level was similar to that directed by the CaMV 35S promoter. A chimeric transcription factor composed of the Gal4 DNA binding domain and an E. coli DNA fragment-encoded activation domain also activated expression.
A chimeric transcription factor composed of the Gal4 DNA binding domain and the proline-rich activation domain of GBF1, a G-box binding transcription factor of Arabidopsis thaliana activated expression of a luciferase reporter gene with Gal4 binding sites in the promoter when tested in a transient assay using soybean cell culture protoplasts (Schindler et al., 1992 EMBO J 11 p 1275-1289). Activated expression was less than that conferred by the Gal4 /E. coli activation domain factor. No activation by the intact Gal4 protein was observed, which was speculated to be due to possible inefficient translation or protein instability.
A chimeric transcription factor composed of the Gal4 DNA binding domain and the activation domain of PvAlf, a seed-specific transcription factor of Phaseolus vulgaris, activated expression of a CAT reporter gene, with Gal4 binding sites in the promoter, in a transient assay in bean cotyledon cells (Bobb et al., 1995 Plant J 8 p 101-113).
No effect of the Gal4 DNA binding domain was detected in stably transformed tobacco plants containing GUS and NPTII reporter genes with Gal4 sites in their promoters (Reuchek et al., 1995 Plant Cell Reports 14 p 773-776). No expression of the Gal4 DNA binding domain protein was detected.
Canadian Patent Application Number 2,150,039, which was published on Aug. 9, 1996, describes a method to control the expression of genes in transgenic plants using native Gal4 as the transactivator. Specifically, it discloses an example in which a GUS reporter gene with Gal4 sites in its promoter is targeted by Gal4 protein expressed from the constitutive CaMV 35S promoter. These two components were co-introduced into Arabidopsis root cells and leaf tissue growing from callus cultures was assayed. When this leaf material was stained for GUS only the veins were positive for expression of the reporter activity. These results are inconsistent with the expectation of constitutive expression throughout the leaf and in all tissues. In other words, the expected result was that the expression of GUS should have been indistinguishable from the example of GUS expressed directly from the 35S promoter (Benfey et al., EMBO J 9:1685-1696 (1990); Odell et al., Nature 313: 810-812 (1985)). Thus, it is likely that the expression which was detected only in the veins is due not to the finctioning of the described system controlled by the 35S promoter. Expression in the veins may be due to integration of GUS adjacent to an endogenous regulatory sequence that directs expression in the veins (Sundaresan et al., 1995, Genes & Development 9:1797-1810; Topping et al., 1994 Plant J. 5:895-903), or to background expression from the promoter operably linked to the GUS coding region. There is no indication that the Gal4 transactivator is expressed and functioning to regulate GUS expression, since it is co-introduced and not added separately to activate the Gal4 site promoter. This result indicates the failure of the transactivation system with Gal4, as was found by other researchers, as discussed above.
While it is known that Gal4 chimeric transcription factor/target promoter systems can function in plant cells in transient assays, there are only reports of failed or inconclusive attempts to use this type of system in stably transformed plants. Thus, there is still a need for a two component system to regulate transgene expression. If such a system was available, then other techniques such as those described in PCT Application having International Publication Number WO 92/08341 which was published on May 29, 1992, the disclosure of which is hereby incorporated by reference, could be used to induce expression of the trait gene only in the production field. In other cases, a two-component system may be used to amplify the expression level from a promoter with desirable tissue or cell type specificity, while maintaining the desired tissue or cell type specificity. Also a two-component system may be used to coordinately regulate multiple transgenes. Still further, an expression system that achieves levels of expression not previously obtained with known promoters is desirable for traits wherein high levels of protein products are required to achieve the traits such as the expression of a protein having a high lysine and/or methionine content which in turn then can improve seed amino acid composition.